Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Summary

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Abstract

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides  mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Introduction

A tight control of protein expression is essential for every living organism to command its own development as well as to react to environmental signals. Hence, a multitude of mechanisms has been invented during evolution to precisely regulate the expression level of each protein encoded by the app. 20,000 genes existing in any eukaryotic cell at any given time of its life. Taking place at different stages of protein production, regulatory mechanisms range from the management of chromatin structure, transcription and RNA handling to the direction of posttranslational protein modifications, transport and degradation.

It is therefore not surp....

Protocol

1. Stock Solutions

1. Resuspend the lyophilized active motif powder in sterile H₂O to a final concentration of 2 µg/µl. Tap carefully for mixing.
2. Prepare small aliquots (e.g., 12 µl each, 2 µl are required per reaction) and store them at -20 °C.

2. Preparation of Cells

1. Seed mammalian cells such as HEK293 cells or primary neurons on a 24 well plate in 500 µl growth medium including antibiotics.
   NOTE: When using neurons, seed the cells at low density and use only the two middle rows of the 24 well plate. Each well will thus have an empty counterpart....

Results

In the following paragraphs, exemplary results illustrating a functional knockdown of a POI (Simiate, for further details please see ^16,17) using Chariot reagent and antibody interference are presented. The findings demonstrate that diminished expression of Simiate impairs transcriptional activity, and, in a dosage dependent manner, induces apoptosis, culminating in mortality rates of over 99% if higher antibody amounts (> 1 µg antibody) are applied (these discoveries were published pr.......

Discussion

Here, we present a protocol to study the significance of protein expression levels in controlling cellular functions in a dose driven manner. The protocol described allows for a fine-tuned manipulation of protein expression in various mammalian cell types, including hippocampal neurons, hence facilitating detailed studies of protein function on the cellular level.

Although RNA interference represents a well known strategy to down-regulate POIs, it has its disadvantages (cp. ¹⁴). Not.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chariot	Active Motif	30025	store at -20 °C
Neurobasal medium	Life technologies	21103-049	warm up to 37 °C before using
1x B27	Life technologies	17504044	store at -20 °C
L-glutamine	Life technologies	25030-149	store at -20 °C
Penicillin and Streptomycin	Life technologies	15140-122	store at -20 °C
Imaris software	Bitplane	n.a.	expensive, but unmatched
Laser Scanning Microscope	Zeiss	n.a.